TCL1 Expression in Chronic Lymphocytic Leukemia (CLL) Regulated by MIR-29 and MIR-181

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis, prognosis and treatment of chronic lymphocytic leukemia (CLL). The invention also provides methods of identifying anti-CLL agents.

GOVERNMENT SUPPORT

This invention was supported, in whole or in part, by grants from NIHGrant/Contract Number PO1 CA81534. The Government has certain rights inthis invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

N/A

BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (B-CLL) is the most common human leukemiain the world accounting for approximately 10,000 new cases each year inthe United States.¹ The TCL1 (T-cell leukemia/lymphoma 1) oncogene wasdiscovered as a target of frequent chromosomal rearrangements at 14q31.2in mature T-cell leukemias.² Previously it was reported that transgenicmice expressing TCL1 in B-cells develop B-CLL.³ The inventor herein nowbelieves that deregulation of TCL1 may be a causal event in thepathogenesis of B-CLL since the inventor has now also shown that TCL1 isa co-activator of the Akt oncoprotein, a critical molecule in thetransduction of anti-apoptotic signals in B- and T-cells.⁴

A recent report suggested that high TCL1 expression in human CLLcorrelates with unmutated V_(H) status and ZAP70 positivity suggestingthat TCL1-driven CLL is an aggressive form of B-CLL.⁵ One of the mostsignificant genetic factors associated with poor prognosis in humanB-CLL is the chromosome 11q deletion.⁶.

MicroRNAs are a large family of highly conserved non-coding genesthought to be involved in temporal and tissue specific gene regulation.⁷We recently demonstrated that microRNA expression profiles can be usedto distinguish normal B-cells from malignant B-CLL cells and thatmicroRNA signatures are associated with prognosis and progression ofchronic lymphocytic leukemia.^(8,9)

No universally successful method for the treatment or prevention ofB-CLL is currently available. The course of treatment for is oftenselected based on a variety of prognostic parameters including ananalysis of specific tumor markers.

In spite of considerable research into therapies for B-CLL, CLL remainsdifficult to diagnose and treat effectively, and the mortality observedin patients indicates that improvements are needed in the diagnosis,treatment and prevention of the disease.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification of achronic lymphocytic leukemia cancer-specific signature of miRNAs thatare differentially-expressed in breast cancer cells, relative to normalcontrol cells.

Accordingly, the invention encompasses methods of diagnosing whether asubject has, or is at risk for developing, chronic lymphocytic leukemia(B-CLL), comprising measuring the level of at least one miR gene productin a test sample from said subject, wherein an alteration in the levelof the miR gene product in the test sample, relative to the level of acorresponding miR gene product in a control sample, is indicative of thesubject either having, or being at risk for developing, B-CLL.

In certain embodiments, at least one miR gene product is miR-29 ormiR-181. In certain embodiments, the at least one miR gene product ismiR-29b and/or miR-181b.

The level of the at least one miR gene product can be measured using avariety of techniques that are well known to those of skill in the art.In one embodiment, the level of the at least one miR gene product ismeasured using Northern blot analysis. In another embodiment, the levelof the at least one miR gene product in the test sample is less than thelevel of the corresponding miR gene product in the control sample. Also,in another embodiment, the level of the at least one miR gene product inthe test sample can be greater than the level of the corresponding miRgene product in the control sample.

The invention also provides methods of diagnosing a B-CLL associatedwith one or more prognostic markers in a subject, comprising measuringthe level of at least one miR gene product in a B-CLL sample from saidsubject, wherein an alteration in the level of the at least one miR geneproduct in the test sample, relative to the level of a corresponding miRgene product in a control sample, is indicative of the subject having aB-CLL associated with the one or more prognostic markers. In oneembodiment, the level of the at least one miR gene product is measuredby reverse transcribing RNA from a test sample obtained from the subjectto provide a set of target oligodeoxynucleotides; hybridizing the targetoligodeoxynucleotides to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the test sample;and, comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample. An alteration in the signal ofat least one miRNA is indicative of the subject either having, or beingat risk for developing, B-CLL.

The invention also encompasses methods of treating CLL in a subject,wherein the signal of at least one miRNA, relative to the signalgenerated from the control sample, is de-regulated (e.g.,down-regulated, up-regulated).

In certain embodiments, a microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR-29 or miR-181 and combinations thereof.

The invention also encompasses methods of diagnosing whether a subjecthas, or is at risk for developing, a B-CLL associated with one or moreadverse prognostic markers in a subject, by reverse transcribing RNAfrom a test sample obtained from the subject to provide a set of targetoligodeoxynucleotides; hybridizing the target oligodeoxynucleotides to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for said test sample; and, comparing the testsample hybridization profile to a hybridization profile generated from acontrol sample. An alteration in the signal is indicative of the subjecteither having, or being at risk for developing, the cancer.

The invention also encompasses methods of treating B-CLL in a subjectwho has a B-CLL in which at least one miR gene product is down-regulatedor up-regulated in the cancer cells of the subject relative to controlcells. When the at least one miR gene product is down-regulated in thecancer cells, the method comprises administering to the subject aneffective amount of at least one isolated miR gene product, such thatproliferation of cancer cells in the subject is inhibited. When the atleast one miR gene product is up-regulated in the cancer cells, themethod comprises administering to the subject an effective amount of atleast one compound for inhibiting expression of the at least one miRgene product, such that proliferation of cancer cells in the subject isinhibited. In certain embodiments, the at least one isolated miR geneproduct is selected miR-29, miR-181 and combinations thereof.

In related embodiments, the invention provides methods of treating B-CLLin a subject, comprising: determining the amount of at least one miRgene product in B-CLL cells, relative to control cells; and altering theamount of miR gene product expressed in the B-CLL cells by:administering to the subject an effective amount of at least oneisolated miR gene product, if the amount of the miR gene productexpressed in the cancer cells is less than the amount of the miR geneproduct expressed in control cells; or administering to the subject aneffective amount of at least one compound for inhibiting expression ofthe at least one miR gene product, if the amount of the miR gene productexpressed in the cancer cells is greater than the amount of the miR geneproduct expressed in control cells, such that proliferation of cancercells in the subject is inhibited. In certain embodiments, at least oneisolated miR gene product is selected from the group consisting ofmiR-29, miR-181, and combinations thereof.

The invention further provides pharmaceutical compositions for treatingB-CLL, comprising at least one isolated miR gene product and apharmaceutically-acceptable carrier. In a particular embodiment, thepharmaceutical compositions the at least one isolated miR gene productcorresponds to a miR gene product that is down-regulated in B-CLL cellsrelative to suitable control cells. In particular embodiments, thepharmaceutical composition is selected from the group consisting ofmiR-29, miR-181 and combinations thereof. In another particularembodiment, the pharmaceutical composition comprises at least one miRexpression inhibitor compound and a pharmaceutically-acceptable carrier.Also, in a particular embodiment, the pharmaceutical compositioncomprises at least one miR expression inhibitor compound is specific fora miR gene product that is up-regulated in B-CLL cells relative tosuitable control cells.

In other embodiments, the present invention provides methods ofidentifying an anti-B-CLL agent, comprising providing a test agent to acell and measuring the level of at least one miR gene product associatedwith decreased expression levels in B-CLL cells, wherein an increase inthe level of the miR gene product in the cell, relative to a suitablecontrol cell, is indicative of the test agent being an anti-B-CLL agent.In certain embodiments, the miR gene product is selected from the groupconsisting of miR-29, miR-181 and combinations thereof.

The present invention also provides methods of identifying an anti-B-CLLagent, comprising providing a test agent to a cell and measuring thelevel of at least one miR gene product associated with increasedexpression levels in B-CLL cells, wherein an decrease in the level ofthe miR gene product in the cell, relative to a suitable control cell,is indicative of the test agent being an anti-B-CLL agent. In aparticular embodiment, the miR gene product is selected from the groupconsisting of miR-29, miR-181 and combinations thereof.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1 f—TCL1 expression is regulated by miR29 and miR18:

FIG. 1 a—TCL1 expression on CLL. Lanes 1-8, CLL samples. Lanes 2 and 6:TCL1 expression was rated as low. All other lines TCL1 expression wasrated as high to very high.

FIG. 1 b—TCL1 expression in three groups of B-CLL. Bars representrelative number of indicated B-CLL samples.

FIG. 1 c—Sequence alignment of miR-29b and miR-181b and 3′ UTR of TCL1.

FIG. 1 d—miR-29 and miR-181 target TCL1 expression in luciferase assays.For miR-29 luciferase assays a fragment of TCL1 cDNA including a regioncomplimentary to miR-29 (Tcl1) was inserted using the XbaI siteimmediately downstream from the stop codon of luciferase into pGL3vector (Promega, Madison, Wis.) construct containing or pGL3 vectoralone as indicated. For miR-181 assays full length TCL1 cDNA wasinserted into pGL3 vector in sense (Tcl1FL) or anti sense (Tcl1FLAS)orientation. 293 cells were co-transfected with the miR-29b or scramblenegative control, as indicated, and pGL3 construct containing a part ofTCL1 cDNA including a region homologous to miR-29 (Tcl1) or pGL3 vectoralone as indicated. For miR-181 assays TCL1FL or TCL1FLAS wereco-transfected with miR-181. Firefly and renilla luciferase activitieswere assayed with the dual luciferase assay system (Promega) and fireflyluciferase activity was normalized to renilla luciferase activity, assuggested by manufacturer. All experiments were carried out intriplicate.

FIG. 1 e—Effect of miR-29b and miR-181b on TCL1 protein expression. 293cells were transfected with pcDNA3TCL1fl (a mammalian expression vectorcontaining full length TCL1 cDNA) alone (lane 1) or co-transfected withpcDNA3TCL1fl and miR-29b (lane 2) pre-miR negative control (lane 3) ormiR-181b (lane 4). TCL1 expression was detected by Western blot usinganti-TCL1 antibody.

FIG. 1 f—Correlation of TCL1 protein expression with miR-181b andmiR-29b by microarray. The values represent microRNA microarrayhybridization signal.

FIGS. 2 a and 2 b—Real time RT-PCR analysis of representative CLLsamples. Three samples with high expression (25, 37 and 41) and foursamples with low expression (55, 56, 72 and 81) of both miR-181 andmiR-29 were chosen. Real time RT-PCR analysis (ABI) was carried out formiR-181a, miR-181b, miR-181c, miR-181d, miR-29a, miR-29b and miR-29caccording to manufacturer's protocol. All experiments were carried outin triplicate.

FIG. 3 contains Table 1 showing the statistically significant microRNAsdifferentiating CLL subtypes.

FIGS. 4 a-4 d contain CLL sample information: FIG. 4 a contains CLLSample Information; FIG. 4 b contains aggressive CLL information; FIG. 4c contains indolent CLL information; and FIG. 4 d contains aggressiveCLL with 11q del. The measurement of the mutational status of theexpressed IgV_(H) genes and immunophenotyping for ZAP-70 was performedas previously described (Rassenti L Z, Huynh L, Toy T L, et al. N Engl.J. Med. 2004; 351:893-901). FISH was performed using the conventionalVysis probes for the CLL panel. These FISH assays can detect thefollowing chromosome anomalies (sets of probes): 11q- and 17p- (ATM at11q23 and P53 at 17p13.1), 13q- and trisomy 12 (D13S319 at 13q14, LAMP1at 13q34 and D12Z3 at centromere 12.

FIG. 5 contains Table 2 showing the pairwise comparison microRNAexpression in three types of B-CLL.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The current invention demonstrates that deregulation of the TCL1oncogene is a causal event in the pathogenesis of the aggressive form ofthis disease, as was verified by using animal models. To study themechanism of TCL1 regulation in CLL, out microRNA expression profilingof three types of CLL was carried out: indolent CLL, aggressive CLL andaggressive CLL showing 11q deletion. Distinct microRNA signaturescorresponding to each group of CLL were identified. It was furtherdetermined that TCL1 expression is regulated by miR-29 and miR-181, twomicroRNAs differentially expressed in CLL. Expression levels of miR-29and miR-181 generally inversely correlated with TCL1 expression in CLLsamples that were examined. It is shown herein that TCL1 expression inCLL is, at least in part, regulated by miR-29 and miR-181 and that thesemiRNAs may be candidates for therapeutic agents in CLLs overexpressingTCL1.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”or “miRNA” refers to the unprocessed or processed RNA transcript from anmiR gene. As the miR gene products are not translated into protein, theterm “miR gene products” does not include proteins. The unprocessed miRgene transcript is also called an “miR precursor,” and typicallycomprises an RNA transcript of about 70-100 nucleotides in length. ThemiR precursor can be processed by digestion with an RNAse (for example,Dicer, Argonaut, or RNAse III, e.g., E. coli RNAse III)) into an active19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA moleculeis also called the “processed” miR gene transcript or “mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAase III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without havingbeen processed from the miR precursor.

The present invention encompasses methods of diagnosing whether asubject has, or is at risk for developing, CLL, comprising measuring thelevel of at least one miR gene product in a test sample from the subjectand comparing the level of the miR gene product in the test sample tothe level of a corresponding miR gene product in a control sample. Asused herein, a “subject” can be any mammal that has, or is suspected ofhaving, breast cancer. In a particular embodiment, the subject is ahuman who has, or is suspected of having, CLL

The level of at least one miR gene product can be measured in cells of abiological sample obtained from the subject. For example, a tissuesample can be removed from a subject suspected of having CLL associatedwith by conventional biopsy techniques. In another example, a bloodsample can be removed from the subject, and white blood cells can beisolated for DNA extraction by standard techniques. The blood or tissuesample is preferably obtained from the subject prior to initiation ofradiotherapy, chemotherapy or other therapeutic treatment. Acorresponding control tissue or blood sample can be obtained fromunaffected tissues of the subject, from a normal human individual orpopulation of normal individuals, or from cultured cells correspondingto the majority of cells in the subject's sample. The control tissue orblood sample is then processed along with the sample from the subject,so that the levels of miR gene product produced from a given miR gene incells from the subject's sample can be compared to the corresponding miRgene product levels from cells of the control sample.

An alteration (i.e., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of CLL in the subject. In one embodiment, the level ofthe at least one miR gene product in the test sample is greater than thelevel of the corresponding miR gene product in the control sample (i.e.,expression of the miR gene product is “up-regulated”). As used herein,expression of an miR gene product is “up-regulated” when the amount ofmiR gene product in a cell or tissue sample from a subject is greaterthan the amount the same gene product in a control cell or tissuesample. In another embodiment, the level of the at least one miR geneproduct in the test sample is less than the level of the correspondingmiR gene product in the control sample (i.e., expression of the miR geneproduct is “down-regulated”). As used herein, expression of an miR geneis “down-regulated” when the amount of miR gene product produced fromthat gene in a cell or tissue sample from a subject is less than theamount produced from the same gene in a control cell or tissue sample.The relative miR gene expression in the control and normal samples canbe determined with respect to one or more RNA expression standards. Thestandards can comprise, for example, a zero miR gene expression level,the miR gene expression level in a standard cell line, or the averagelevel of miR gene expression previously obtained for a population ofnormal human controls.

The level of a miR gene product in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques for determining RNA expressionlevels in cells from a biological sample (e.g., Northern blot analysis,RT-PCR, in situ hybridization) are well known to those of skill in theart. In a particular embodiment, the level of at least one miR geneproduct is detected using Northern blot analysis. For example, totalcellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapter 7, the entire disclosure of whichis incorporated by reference.

Suitable probes for Northern blot hybridization of a given miR geneproduct can be produced from the nucleic acid sequences of the givenmiR. Methods for preparation of labeled DNA and RNA probes, and theconditions for hybridization thereof to target nucleotide sequences, aredescribed in Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters10 and 11, the disclosures of which are incorporated herein byreference.

For example, the nucleic acid probe can be labeled with, e.g., aradionuclide, such as ³H, ³²P, ³³P, ¹⁴C, or ³⁵S; a heavy metal; or aligand capable of functioning as a specific binding pair member for alabeled ligand (e.g., biotin, avidin or an antibody), a fluorescentmolecule, a chemiluminescent molecule, an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference. Suitable probes for in situhybridization of a given miR gene product can be produced from thenucleic acid sequences.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR). The levels of miR gene transcripts can be quantifiedin comparison with an internal standard, for example, the level of mRNAfrom a “housekeeping” gene present in the same sample. A suitable“housekeeping” gene for use as an internal standard includes, e.g.,myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The methodsfor quantitative RT-PCR and variations thereof are within the skill inthe art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer. Assessing cancer-specific expression levels for hundredsof miR genes is time consuming and requires a large amount of total RNA(at least 20 μg for each Northern blot) and autoradiographic techniquesthat require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set of probeoligodeoxynucleotides that are specific for a set of miR genes. Usingsuch a microarray, the expression level of multiple microRNAs in abiological sample can be determined by reverse transcribing the RNAs togenerate a set of target oligodeoxynucleotides, and hybridizing them toprobe oligodeoxynucleotides on the microarray to generate ahybridization, or expression, profile. The hybridization profile of thetest sample can then be compared to that of a control sample todetermine which microRNAs have an altered expression level in CLL. Asused herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. “Target oligonucleotide” or “targetoligodeoxynucleotide” refers to a molecule to be detected (e.g., viahybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for an miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal cells may be distinguished from CLL cells, and within CLLcells, different prognosis states (good or poor long term survivalprospects, for example) may be determined. By comparing expressionprofiles of CLL cells in different states, information regarding whichgenes are important (including both up- and down-regulation of genes) ineach of these states is obtained. The identification of sequences thatare differentially expressed in CLL cells or normal cells, as well asdifferential expression resulting in different prognostic outcomes,allows the use of this information in a number of ways. For example, aparticular treatment regime may be evaluated (e.g., to determine whethera chemotherapeutic drug act to improve the long-term prognosis in aparticular patient). Similarly, diagnosis may be done or confirmed bycomparing patient samples with the known expression profiles.Furthermore, these gene expression profiles (or individual genes) allowscreening of drug candidates that suppress the CLL expression profile orconvert a poor prognosis profile to a better prognosis profile.

Accordingly, the invention provides methods of diagnosing whether asubject has, or is at risk for developing, CLL, comprising reversetranscribing RNA from a test sample obtained from the subject to providea set of target oligo-deoxynucleotides, hybridizing the targetoligo-deoxynucleotides to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the test sample,and comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample, wherein an alteration in thesignal of at least one miRNA is indicative of the subject either having,or being at risk for developing, CLL.

In one embodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of the human miRNome. In aparticular embodiment, the microarray comprises miRNA-specific probeoligo-nucleotides for one or more miRNAs selected from the groupconsisting of miR-29 or miR-181 and combinations thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs from both species may also be printed on the microchip, providingan internal, relatively stable, positive control for specifichybridization. One or more appropriate controls for non-specifichybridization may also be included on the microchip. For this purpose,sequences are selected based upon the absence of any homology with anyknown miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT at 37°C. for 40 minutes. At positions on the array where the immobilized probeDNA recognizes a complementary target cDNA in the sample, hybridizationoccurs. The labeled target cDNA marks the exact position on the arraywhere binding occurs, allowing automatic detection and quantification.The output consists of a list of hybridization events, indicating therelative abundance of specific cDNA sequences, and therefore therelative abundance of the corresponding complementary miRs, in thepatient sample. According to one embodiment, the labeled cDNA oligomeris a biotin-labeled cDNA, prepared from a biotin-labeled primer. Themicroarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool would allow for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a cancer (e.g.,CLL) is quantitatively reverse transcribed to provide a set of labeledtarget oligodeoxynucleotides complementary to the RNA in the sample. Thetarget oligodeoxynucleotides are then hybridized to a microarraycomprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal). More preferably, the profile recorded includes the intensity ofthe signal from each hybridization. The profile is compared to thehybridization profile generated from a normal, i.e., noncancerous,control sample. An alteration in the signal is indicative of thepresence of the cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

The invention also provides methods of diagnosing a CLL associated withone or more prognostic markers, comprising measuring the level of atleast one miR gene product in a CLL test sample from a subject andcomparing the level of the at least one miR gene product in the CLL testsample to the level of a corresponding miR gene product in a controlsample. An alteration (e.g., an increase, a decrease) in the signal ofat least one miRNA in the test sample relative to the control sample isindicative of the subject either having, or being at risk fordeveloping, CLL associated with the one or more prognostic markers.

The CLL can be associated with one or more prognostic markers orfeatures, including, a marker associated with an adverse (i.e.,negative) prognosis, or a marker associated with a good (i.e., positive)prognosis. In certain embodiments, the CLL that is diagnosed using themethods described herein is associated with one or more adverseprognostic features.

Particular microRNAs whose expression is altered in CLL cells associatedwith each of these prognostic markers are described herein. In oneembodiment, the level of the at least one miR gene product is measuredby reverse transcribing RNA from a test sample obtained from the subjectto provide a set of target oligodeoxynucleotides, hybridizing the targetoligodeoxynucleotides to a microarray that comprises miRNA-specificprobe oligonucleotides to provide a hybridization profile for the testsample, and comparing the test sample hybridization profile to ahybridization profile generated from a control sample.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to the formation of CLL. Therefore, altering thelevel of the miR gene product (e.g., by decreasing the level of a miRthat is up-regulated in CLL cells, by increasing the level of a miR thatis down-regulated in cancer cells) may successfully treat the CLL.Examples of putative gene targets for miRNAs that are deregulated in CLLcells are described herein.

Accordingly, the present invention encompasses methods of treating CLLin a subject, wherein at least one miR gene product is de-regulated(e.g., down-regulated, up-regulated) in the cancer cells of the subject.When the at least one isolated miR gene product is down-regulated in theCLL cells, the method comprises administering an effective amount of theat least one isolated miR gene product such that proliferation of cancercells in the subject is inhibited. When the at least one isolated miRgene product is up-regulated in the cancer cells, the method comprisesadministering to the subject an effective amount of at least onecompound for inhibiting expression of the at least one miR gene,referred to herein as miR gene expression inhibition compounds, suchthat proliferation of CLL cells is inhibited.

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, CLL, including preventing or delaying the onset of the diseasesymptoms, and/or lessening the severity or frequency of symptoms of thedisease or condition. The terms “subject” and “individual” are definedherein to include animals, such as mammals, including but not limitedto, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guineapigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent,or murine species. In a preferred embodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of a cancer cell in asubject suffering from CLL. One skilled in the art can readily determinean effective amount of an miR gene product to be administered to a givensubject, by taking into account factors, such as the size and weight ofthe subject; the extent of disease penetration; the age, health and sexof the subject; the route of administration; and whether theadministration is regional or systemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate or estimated body weight of a subject to betreated. Preferably, such effective amounts are administeredparenterally or enterally, as described herein. For example, aneffective amount of the isolated miR gene product is administered to asubject can range from about 5-3000 micrograms/kg of body weight, fromabout 700-1000 micrograms/kg of body weight, or greater than about 1000micrograms/kg of body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, an miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, an miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, an miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one which issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or an miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in substantially-purified form, or can exist in acell into which the miR gene product has been delivered. Thus, an miRgene product which is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. An miR gene productproduced inside a cell from an miR precursor molecule is also consideredto be “isolated” molecule.

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cancercells.

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products which are expressed from recombinantplasmids can also be delivered to, and expressed directly in, the cancercells. The use of recombinant plasmids to deliver the miR gene productsto cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which are incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich are incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cancer cells. The use ofrecombinant viral vectors to deliver the miR gene products to cancercells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, for example, the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant viral vectors of the invention can also compriseinducible or regulatable promoters for expression of the miR geneproducts in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therap.2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therap. 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding an miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound which inhibits miR expressioncan also be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the active, mature form of miRgene product after treatment is less than the amount produced prior totreatment. One skilled in the art can readily determine whether miRexpression has been inhibited in a cancer cell, using for example thetechniques for determining miR transcript level discussed above for thediagnostic method. Inhibition can occur at the level of gene expression(i.e., by inhibiting transcription of a miR gene encoding the miR geneproduct) or at the level of processing (e.g., by inhibiting processingof a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of a cancercell in a subject suffering from a cancer associated with acancer-associated chromosomal feature. One skilled in the art canreadily determine an effective amount of an miR expression-inhibitingcompound to be administered to a given subject, by taking into accountfactors, such as the size and weight of the subject; the extent ofdisease penetration; the age, health and sex of the subject; the routeof administration; and whether the administration is regional orsystemic.

For example, an effective amount of the expression-inhibiting compoundcan be based on the approximate or estimated body weight of a subject tobe treated. Such effective amounts are administered parenterally orenterally, among others, as described herein. For example, an effectiveamount of the expression-inhibiting compound administered to a subjectcan range from about {tilde over (5)}-3000 micrograms/kg of body weight,from about 700-1000 micrograms/kg of body weight, or it can be greaterthan about 1000 micrograms/kg of body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject. For example, an expression-inhibiting compound can beadministered to the subject once (e.g., as a single injection ordeposition). Alternatively, an expression-inhibiting compound can beadministered once or twice daily to a subject for a period of from aboutthree to about twenty-eight days, more preferably from about seven toabout ten days. In a particular dosage regimen, an expression-inhibitingcompound is administered once a day for seven days. Where a dosageregimen comprises multiple administrations, it is understood that theeffective amount of the expression-inhibiting compound administered tothe subject can comprise the total amount of compound administered overthe entire dosage regimen.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such asribozymes. Each of these compounds can be targeted to a given miR geneproduct and destroy or induce the destruction of the target miR geneproduct.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99% or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence whichis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofwhich are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA orRNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequencecomplementary to a contiguous nucleic acid sequence in an miR geneproduct. The antisense nucleic acid can comprise a nucleic acid sequencethat is 50-100% complementary, 75-100% complementary, or 95-100%complementary to a contiguous nucleic acid sequence in an miR geneproduct. Nucleic acid sequences for the miR gene products are providedherein. Without wishing to be bound by any theory, it is believed thatthe antisense nucleic acids activate RNase H or another cellularnuclease that digests the miR gene product/antisense nucleic acidduplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of an miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in an miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid foruse in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al.(1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No.4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cancer cells in a subject who has a cancer associated with acancer-associated chromosomal feature. As used herein, to “inhibit theproliferation of a cancer cell” means to kill the cell, or permanentlyor temporarily arrest or slow the growth of the cell. Inhibition ofcancer cell proliferation can be inferred if the number of such cells inthe subject remains constant or decreases after administration of themiR gene products or miR gene expression-inhibiting compounds. Aninhibition of cancer cell proliferation can also be inferred if theabsolute number of such cells increases, but the rate of tumor growthdecreases.

The number of cancer cells in a subject's body can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The miR gene products or miR gene expression-inhibiting compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cancer cells of the subject. For example, the miR geneproducts or miR expression inhibiting compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds. Inone embodiment, the cells are transfected with a plasmid or viral vectorcomprising sequences encoding at least one miR gene product or miR geneexpression inhibiting compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/10⁵ cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 10⁵ cells can be used.

An miR gene product or miR gene expression inhibiting compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and intravenous administration into the patient.

In the present methods, an miR gene product or miR gene productexpression inhibiting compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or expression inhibiting compound.Suitable delivery reagents include, e.g., the Mirus Transit TKOlipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations(e.g., polylysine), and liposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression inhibiting compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein.

In a particular embodiment, liposomes are used to deliver an miR geneproduct or miR gene expression-inhibiting compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands which bindto receptors prevalent in cancer cells, such as monoclonal antibodiesthat bind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both opsonization-inhibition moieties anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers, such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM1. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups. Preferably, the opsonization-inhibiting moiety is aPEG, PPG, or derivatives thereof. Liposomes modified with PEG orPEG-derivatives are sometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example solid tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating CLL. In one embodiment, thepharmaceutical compositions comprise at least one isolated miR geneproduct and a pharmaceutically-acceptable carrier. In a particularembodiment, the at least one miR gene product corresponds to a miR geneproduct that has a decreased level of expression in CLL cells relativeto suitable control cells. In certain embodiments the isolated miR geneproduct is selected from the group consisting of miR-29 or miR-181 andcombinations thereof.

In other embodiments, the pharmaceutical compositions of the inventioncomprise at least one miR expression inhibition compound. In aparticular embodiment, the at least one miR gene expression inhibitioncompound is specific for a miR gene whose expression is greater in CLLcells than control cells. In certain embodiments, the miR geneexpression inhibition compound is specific for one or more miR geneproducts selected from the group consisting of consisting of miR-29 ormiR-181 and combinations thereof.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical formulations” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical formulations comprise at least one miR geneproduct or miR gene expression inhibition compound (or at least onenucleic acid comprising sequences encoding them) (e.g., 0.1 to 90% byweight), or a physiologically acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. The pharmaceutical formulations ofthe invention can also comprise at least one miR gene product or miRgene expression inhibition compound (or at least one nucleic acidcomprising sequences encoding them) which are encapsulated by liposomesand a pharmaceutically-acceptable carrier.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR gene expressioninhibition compound (or at least one nucleic acid comprising sequencesencoding them) which is resistant to degradation by nucleases. Oneskilled in the art can readily synthesize nucleic acids which arenuclease resistant, for example by incorporating one or moreribonucleotides that are modified at the 2′-position into the miR geneproducts. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy, and O-allyl.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression inhibition compound (or at least one nucleic acidcomprising sequences encoding them) encapsulated in a liposome asdescribed above, and a propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

The invention also encompasses methods of identifying an anti-CLL agent,comprising providing a test agent to a cell and measuring the level ofat least one miR gene product in the cell. In one embodiment, the methodcomprises providing a test agent to a cell and measuring the level of atleast one miR gene product associated with decreased expression levelsin CLL cells. An increase in the level of the miR gene product in thecell, relative to a suitable control cell, is indicative of the testagent being an anti-CLL agent. In a particular embodiment, at least onemiR gene product associated with decreased expression levels in CLLcells is selected from the group consisting of miR-29 or miR-181 andcombinations thereof.

In other embodiments the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in CLL cells. A decrease in the levelof the miR gene product in the cell, relative to a suitable controlcell, is indicative of the test agent being an anti-CLL agent. In aparticular embodiment, at least one miR gene product associated withincreased expression levels in CLL cells is selected from the groupconsisting of miR-29 or miR-181 and combinations thereof.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art.

The invention will now be illustrated by the following non-limitingexamples.

Examples

CLL Samples and MicroRNA Microchip Experiments.

Eighty CLL samples were obtained after informed consent from patientsdiagnosed with CLL at the CLL Research Consortium institutions. Briefly,blood was obtained from CLL patients, lymphocytes were isolated throughFicoll/Hypaque gradient centrifugation (Amersham, Piscataway, N.J.) andprocessed for RNA extraction using the standard Trizol method. Proteinextraction was carried out as described previously.¹⁰ MicroRNA-microchipexperiments were performed as previously described.⁹ Each microRNAmicrochip contained duplicates probes, corresponding to 326 human and249 mouse microRNA genes. Statistical analysis was carried out aspreviously described.¹¹ To identify statistically significantdifferentially expressed microRNA, class prediction analyses wereperformed using BRB ArrayTools.

DNA-Constructs, Transfection, Western Blotting and Luciferase Assay.

Full length TCL1 cDNA including 5′ and 3′ UTRs cDNA was cloned into apUSEamp vector (Upstate Biotechnology, Chicago, Ill.) (used in FIG. 1e). MiR-29b and miR-181b RNA duplexes were purchased from Ambion(Austin, Tex.). Transfections were carried out as previouslydescribed.¹² Firefly and renilla luciferase activities were assayed withthe dual luciferase assay system (Promega) and firefly luciferaseactivity was normalized to renilla luciferase activity. Cell lysatepreparations and Western blot analysis were carried out using anti-TCL1monoclonal antibody (clone 27D6) as described previously.⁴

Results and Discussion.

High Expression of Tcl1 Correlates with Aggressive B-CLL Phenotype.

To evaluate TCL1 and microRNA expression in B-CLL samples, three groupsof B-CLL were chosen: 23 samples of indolent B-CLL, 25 samples ofaggressive B-CLL and 32 samples of aggressive B-CLL showing 11qdeletion. Detailed descriptions of the samples are shown in FIGS. 4 a-4d.

MicroRNA microchip experiments revealed that three groups of CLL showsignificant characteristic differences in microRNA expression pattern(see FIG. 3—Table 1 and FIG. 5—Table 2).

To determine TCL1 protein expression in three groups of CLL, Westernblot analysis using 27D6 TCL1 monoclonal antibody was carried out.Results of these experiments are shown on FIGS. 1 a-b.

TCL1 expression was assessed as low, medium, high and very high. Ourexperiments revealed low levels in 15 of 23 (65%) indolent B-CLLs, in 11of 25 (44%) aggressive B-CLLs and in 1 of 32 (3%) aggressive B-CLLs with11q deletions; whereas high and very high TCL1 expression was observedin 1 of 23 (4%) indolent B-CLLs, in 14 of 25 (56%) aggressive B-CLLs andin 24 of 32 (75%) aggressive B-CLLs with 11q deletions (FIG. 1 b).

It is believed by the inventor herein that TCL1 overexpressioncorrelates with aggressive B-CLL phenotype (p<10⁻⁶) and 11q deletions(p<10⁻⁴). The results are consistent with the recently published studydemonstrating that high TCL1 expression in human CLL correlates withunmutated V_(H) status and ZAP70 positivity.⁵

MiR-29 and miR-181 Target Tcl1 Expression.

To determine which miRNA(s) target TCL1, RNAhybrid software offered byBielefeld University Bioinformatics Server and miRBase database¹³ wasused. Among miR-candidates targeting TCL1, it was found that miR-29b andmiR-181b (FIG. 1 c, several other sites with lower homology not shown)are also down-regulated in aggressive B-CLLs with 11q deletions (FIG.3—Table 1).

The expression of these miRs was confirmed by real time RT-PCR inrepresentative set of samples (FIGS. 4 a-4 d).

Furthermore, it was previously shown that expression of members ofmiR-29 family could discriminate between CLL samples with good and badprognosis.⁹ It was then determined whether these miRs indeed target TCL1expression using the TCL1 3′ UTR inserted downstream downstream ofluciferase ORF, as previously described.¹² HEK293 cells wereco-transfected with the miR-29b or scramble negative control, asindicated, and pGL3 construct containing a part of TCL1 cDNA including aregion homologous to miR-29 (Tcl1) or pGL3 vector alone as indicated.

For miR-181 assays full length TCL1 cDNA was inserted into pGL3 vectorin sense (Tcl1FL) or anti sense (Tcl1FLAS) orientation. FIG. 1 d showsthat TCL1 mRNA expression is inhibited by miR-29 and miR-181. To confirmthese findings full-length TCL1 cDNA including 5′ and 3′ UTRs werecloned into CMV mammalian expression vector and investigated whethermiR-29b and miR-181b affect TCL1 protein expression levels. Weco-transfected this construct with miR-29b, miR-181b and Pre-miRnegative control (scramble) into 293 cells as indicated on FIG. 1 e.

These experiments revealed that co-expression of TCL1 with miR-29 andmiR-181 significantly decreased TCL1 expression (FIG. 1 e, lane 2 versuslanes 1 and 3).

Thus, it is shown herein that miR-29b and miR-181b target TCL1expression on mRNA and protein levels. Interestingly, there was aninverse correlation between miR-29b and miR-181b expression and TCL1protein expression in B-CLL samples (FIG. 1 f): all samples showing highmiR-29b and miR-181b expression also show low or medium TCL1 expression;all samples showing very high TCL1 expression show mostly low expressionof miR-29b and miR-181b. These results show that TCL1 expression in CLLis, at least in part, regulated by miR-29 and miR-181.

It is demonstrated herein that TCL1 expression is regulated by miR-29and miR-181 and this regulation is relevant to three groups of B-CLLstudied. Although a reverse correlation between TCL1 protein expressionand these two miRs was observed, significant proportion of B-CLL samplesshow low TCL1 expression and low expression of miR-29 and miR-181 (FIG.1 f). This suggests that in these samples TCL1 expression isdown-regulated transcriptionally or by other miRNAs. The fact thatneither miR-29 nor miR-181 is located at 11q suggests that that regionmay contain an important regulator of the expression of these two miRs.

Interestingly, miR-181 is differentially expressed in B-cells and TCL1is mostly B-cell specific gene.¹⁴ While not wishing to be bound bytheory, the inventor herein believes that there is an inversecorrelation between TCL1 and miR-181 expression at different stages ofB-cell maturation. Since miR-29 and miR-181 are natural TCL1 inhibitors,these miRs can be useful candidates for therapeutic agents in B-CLLoverexpressing TCL1.

In accordance with the provisions of the patent statutes, the principleand mode of operation of this invention have been explained andillustrated in its preferred embodiment. However, it must be understoodthat this invention may be practiced otherwise than as specificallyexplained and illustrated without departing from its spirit or scope.

REFERENCES

The references discussed above and the following references, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference.

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2. Virgilio L, Narducci M G, Isobe M, et al. Identification of the TCL1gene involved in T-cell malignancies. Proc Natl. Acad. Sci. USA 1994;91:12530-12534.

3. Bichi R, Shinton S A, Martin E S, et al. Human chronic lymphocyticleukemia modeled in mouse by targeted TCL1 expression. Proc. Natl. Acad.Sci. USA. 2002; 99:6955-6960.

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1. A method of diagnosing whether a subject has, or is at risk fordeveloping, chronic lymphocytic leukemia (B-CLL), comprising measuringthe level of at least one miR gene product in a test sample from saidsubject, wherein an alteration in the level of the miR gene product inthe test sample, relative to the level of a corresponding miR geneproduct in a control sample, is indicative of the subject either having,or being at risk for developing, B-CLL.
 2. The method of claim 1,wherein the at least one miR gene product is miR-29 or miR-181.
 3. Themethod of claim 1, wherein the at least one miR gene product is miR-29b.4. The method of claim 1, wherein the at least one miR gene product ismiR-181b.
 5. The method of claim 1, wherein the level of the at leastone miR gene product is measured using Northern blot analysis.
 6. Themethod of claim 1, wherein the level of the at least one miR geneproduct in the test sample is less than the level of the correspondingmiR gene product in the control sample.
 7. The method of claim 1,wherein the level of the at least one miR gene product in the testsample is greater than the level of the corresponding miR gene productin the control sample.
 8. A method of diagnosing a B-CLL associated withone or more prognostic markers in a subject, comprising measuring thelevel of at least one miR gene product in a B-CLL sample from saidsubject, wherein an alteration in the level of the at least one miR geneproduct in the test sample, relative to the level of a corresponding miRgene product in a control sample, is indicative of the subject having aB-CLL associated with the one or more prognostic markers.
 9. A method ofdiagnosing whether a subject has, or is at risk for developing, B-CLL,comprising: (1) reverse transcribing RNA from a test sample obtainedfrom the subject to provide a set of target oligodeoxynucleotides; (2)hybridizing the target oligodeoxynucleotides to a microarray comprisingmiRNA-specific probe oligonucleotides to provide a hybridization profilefor the test sample; and, (3) comparing the test sample hybridizationprofile to a hybridization profile generated from a control sample,wherein an alteration in the signal of at least one miRNA is indicativeof the subject either having, or being at risk for developing, B-CLL.10. The method of claim 9 wherein the signal of at least one miRNA,relative to the signal generated from the control sample, isdown-regulated.
 11. The method of claim 9 wherein the signal of at leastone miRNA, relative to the signal generated from the control sample, isup-regulated.
 12. The method of claim 9 wherein the microarray comprisesmiRNA-specific probe oligonucleotides for one or more miRNAs selectedfrom the group consisting of miR-29 or miR-181 and combinations thereof.13. A method of diagnosing whether a subject has, or is at risk fordeveloping, a B-CLL associated with one or more adverse prognosticmarkers in a subject, comprising: (1) reverse transcribing RNA from atest sample obtained from the subject to provide a set of targetoligodeoxynucleotides; (2) hybridizing the target oligodeoxynucleotidesto a microarray comprising miRNA-specific probe oligonucleotides toprovide a hybridization profile for said test sample; and, (3) comparingthe test sample hybridization profile to a hybridization profilegenerated from a control sample, wherein an alteration in the signal isindicative of the subject either having, or being at risk fordeveloping, the cancer.
 14. The method of claim 13, wherein themicroarray comprises at least one miRNA-specific probe oligonucleotidefor a miRNA selected from the group consisting of miR-29 or miR-181 andcombinations thereof.
 15. A method of treating B-CLL in a subject whohas a B-CLL in which at least one miR gene product is down-regulated orup-regulated in the cancer cells of the subject relative to controlcells, comprising: (1) when the at least one miR gene product isdown-regulated in the cancer cells, administering to the subject aneffective amount of at least one isolated miR gene product, such thatproliferation of cancer cells in the subject is inhibited; or (2) whenthe at least one miR gene product is up-regulated in the cancer cells,administering to the subject an effective amount of at least onecompound for inhibiting expression of the at least one miR gene product,such that proliferation of cancer cells in the subject is inhibited. 16.The method of claim 15, wherein the at least one isolated miR geneproduct in step (1) is selected miR-29, miR-181 and combinationsthereof.
 17. The method of claim 15, wherein the at least one miR geneproduct in step (2) is selected from the group consisting of miR-291,miR-181 and combinations thereof.
 18. A method of treating B-CLL in asubject, comprising: (1) determining the amount of at least one miR geneproduct in B-CLL cells, relative to control cells; and (2) altering theamount of miR gene product expressed in the B-CLL cells by: i)administering to the subject an effective amount of at least oneisolated miR gene product, if the amount of the miR gene productexpressed in the cancer cells is less than the amount of the miR geneproduct expressed in control cells; or ii) administering to the subjectan effective amount of at least one compound for inhibiting expressionof the at least one miR gene product, if the amount of the miR geneproduct expressed in the cancer cells is greater than the amount of themiR gene product expressed in control cells, such that proliferation ofcancer cells in the subject is inhibited.
 19. The method of claim 18,wherein the at least one isolated miR gene product in step (i) isselected from the group consisting of miR-29, miR-181, and combinationsthereof.
 20. The method of claim 18, wherein the at least one miR geneproduct in step (ii) is selected from the group consisting of miR-29,miR-181, and combinations thereof.
 21. A pharmaceutical composition fortreating B-CLL, comprising at least one isolated miR gene product and apharmaceutically-acceptable carrier.
 22. The pharmaceutical compositionof claim 21, wherein the at least one isolated miR gene productcorresponds to a miR gene product that is down-regulated in B-CLL cellsrelative to suitable control cells.
 23. The pharmaceutical compositionof claim 22, wherein the isolated miR gene product is selected from thegroup consisting of miR-29, miR-181 and combinations thereof.
 24. Apharmaceutical composition for treating B-CLL, comprising at least onemiR expression inhibitor compound and a pharmaceutically-acceptablecarrier.
 25. The pharmaceutical composition of claim 24, wherein the atleast one miR expression inhibitor compound is specific for a miR geneproduct that is up-regulated in B-CLL cells relative to suitable controlcells.
 26. The pharmaceutical composition of claim 25, wherein the atleast one miR expression inhibitor compound is specific for a miR geneproduct selected from the group consisting of miR-29, miR-181 andcombinations thereof.
 27. A method of identifying an anti-B-CLL agent,comprising providing a test agent to a cell and measuring the level ofat least one miR gene product associated with decreased expressionlevels in B-CLL cells, wherein an increase in the level of the miR geneproduct in the cell, relative to a suitable control cell, is indicativeof the test agent being an anti-B-CLL agent.
 28. The method of claim 27,wherein the miR gene product is selected from the group consisting ofmiR-29, miR-181 and combinations thereof.
 29. A method of identifying ananti-B-CLL agent, comprising providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withincreased expression levels in B-CLL cells, wherein an decrease in thelevel of the miR gene product in the cell, relative to a suitablecontrol cell, is indicative of the test agent being an anti-B-CLL agent.30. The method of claim 29, wherein the miR gene product is selectedfrom the group consisting of miR-29, miR-181 and combinations thereof.